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Homologous recombination cloning
Homologous recombination cloning is a molecular genetics technique in which an engineered sequence of DNA is swapped in for a targeted region of a cell’s chromosome. This powerful technique allows researchers to stably express a gene construct in a cell or to knockout a specific endogenous gene by integrating the transforming DNA at a targeted host chromosome location. Background: Homologous recombination during meiosis During gamete formation homologous chromosomes are aligned and then separated into the two forming haplotype daughter cells. While the homologous chromosomes are aligned they can form temporary junctions with each other along sequence lengths that are highly identical. The resulting four intertwined strands of DNA (two from each chromosome involved) are called Holliday Junctions, after Robin Holliday, who first proposed them to explain the observed exchange of genetic information in a fungal parasite. As Holliday Junctions are resolved to allow for the separation of the homologous chromosomes, regions in between the junctions can be exchanged, resulting in a novel combination of genes going to the gametes (1). Technique Homologous recombination cloning utilizes the endogenous homologous recombination machinery of the host cell to alter its genome in a site directed manner. The technique consists of synthesizing a DNA construct through PCR amplification using primers containing ~50 base pairs homologous to the site of insertion followed by ~20 base pairs homologous to the insert DNA. In this way, knowledge of the host cell genome is required to facilitate integration, and to target the exogenous DNA to a particular genetic location. The amplified DNA is then inserted into the cells through an appropriate transformation method. Because the exogenous DNA is linear – and relatively abundant in the cell following transformation – it is likely that among a population of transformed cells, one or more cells will integrate the DNA at the target site. To efficiently screen for those cells that have successfully integrated the DNA, usually a genetic marker, either conveying drug resistance or rescuing auxotrophy, is included in the DNA construct. After using one of these screening methods to select for cells that have taken up the DNA, sequencing of clonal populations is required to confirm integration at the correct sequence (2). Research Applications Homologous recombination cloning provides a practical method to replace an endogenous gene with a mutant variant, or to study the effect of knocking-out a gene by replacing it with a selectable marker. This technique is particularly simple to employ in a single-celled model system, such as the fission yeast, S. pombe. Bahler et al described the technique – including specific plasmid constructs with various selectable markers, purification tags, and GFP – in the journal ,Yeast (1998) (2). Homologous recombination cloning, or gene targeting, is also a tremendously important technique in higher eukaryotes, particular in mice. Mouse embryonic stem cell nuclei are transformed in a similar manner described above, and then inserted into host cell line. The cells then are induced to differentiate and divide, forming a mosaic animal that can pass the knocked-out genotype to its off-spring through normal Mendelian genetics, and selected for in future generations. This has allowed researchers to theoretically create a knockout model for every mammalian gene. Since its development in 1986 by Mario Capecchi and Oliver Smithies, this technique has been used to create more than 10,000 mammalian gene knock-outs, and over 500 human disease models have been developed(3). References 1. San Filippo J, Sung P, & Klein H (2008) Mechanism of eukaryotic homologous recombination. Annual review of biochemistry 77:229-257. 2. Bahler J, et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14(10):943-951. 3. nobelprize.org